This invention relates to extracting quantitative, three-dimensional molecular information from living mammals and patients using fluorochromes and new optical tomographic imaging methods.
Molecular imaging can be broadly defined as the characterization and measurement of biological processes at the cellular and molecular level in mammals and human patients. In contradistinction to xe2x80x9cclassicalxe2x80x9d diagnostic imaging, for example, magnetic resonance (MR), computed tomography (CT), and ultrasound (US) imaging, molecular imaging analyses molecular abnormalities that are the basis of disease, rather than imaging the end effects of these molecular alterations. Specific imaging of molecular targets allows earlier detection and characterization of disease, as well as earlier and direct molecular assessment of treatment efficacy. Molecular imaging can theoretically be performed with different imaging technologies, up to now preferably with nuclear imaging technologies, e.g., PET and SPECT imaging) which have high sensitivity of probe detection. The IV administered imaging probes typically recognize a given target. Alternatively, some probes detectable by MR imaging have been developed (Moats et al., Angewandte Chemie Int. Ed., 36:726-731, 1997; Weissleder et al., Nat. Med., 6:351-5, 2000), although their detection threshold is generally in the micromolar instead of the pico/femptomolar range of isotope probes.
An alternative method is to use fluorescent probes for target recognition. For example, enzyme activatable fluorochrome probes are described in Weissleder et al., U.S. Pat. No. 6,083,486, and fluorescent molecular beacons that become fluorescent after DNA hybridization are described in Tyagi et al., Nat. Biotechnol., 16:49-53, 1998. Fluorescence activatable probes have been used in tissue culture and histologic sections and detected using fluorescence microscopy. When administered in vivo, fluorescence activatable probes have been detected by surface-weighted reflectance imaging (Weissleder et al., Nat. Biotechnol., 17:375-8, 1999); Mahmood et al., Radiology, 213:866-70, 1999. However, imaging in deep tissues ( greater than 5 mm from the surface), in absorbing and scattering media such as mammalian tissues, and quantitating fluorescence (and in particular fluorescence activation) has not been described.
To image light interactions in deeper tissues, light in the near infrared (near-IR or NIR) instead of the visible spectrum is preferred. Imaging with near infrared (near-IR or NIR) light has been in the frontier of research for resolving and quantifying tissue function. Light offers unique contrast mechanisms that can be based on absorption, e.g., probing of hemoglobin concentration or blood saturation, and/or fluorescence, e.g., probing for weak auto-fluorescence, or exogenously administered fluorescent probes (Neri et al., Nat. Biotech., 15:1271-1275, 1997; Ballou et al., Cancer Immunol. Immunother., 41:257-63,1995; and Weissleder, 1999). In either application, NIR photons undergo significant elastic scattering when traveling through tissue. This results in light xe2x80x9cdiffusionxe2x80x9d in tissue that hinders resolution and impairs the ability to produce diagnostically interpretable images using simple xe2x80x9cprojectionxe2x80x9d approaches (transillumination), as in x-ray imaging.
During the last decade, mathematical modeling of light propagation in tissue, combined with technological advancements in photon sources and detection techniques has made possible the application of tomographic principles (Kak and Slaney, xe2x80x9cPrinciples of Computerized Tomographic Imaging,xe2x80x9d IEEE Press, New York, 1988, pp. 208-218); Arridge, Inverse Problems; 15:R41-R93, 1999) for imaging with diffuse light. Diffuse Optical Tomography (DOT) uses multiple projections and deconvolves the scattering effect of tissue. DOT imaging has been used for quantitative, three-dimensional imaging of intrinsic absorption and scattering (see, e.g., Ntziachristos et al., Proc. Natl. Acad. Sci., USA, 97:2767-72, 2000), and also Benaron et al., J. Cerebral Blood Flow Metabol., 20(3):469-77, 2000). These fundamental quantities can be used to derive tissue oxy- and deoxy-hemoglobin concentrations, blood oxygen saturation (Li et al., Appl. Opt., 35:3746-3758, 1996) or hematoma detection in diffuse media.
Although intrinsic-contrast for DOT imaging may be useful in certain situations, e.g., for functional brain activation studies or hematoma detection, these applications do not allow the extraction of highly specific molecular information from living tissues. Fluorochrome concentration has been measured by absorption measurements (Ntziachristos et al., 2000) or by fluroescence measurements in phantoms (Chang et al., IEEE Trans. Med. Imag., 16:68-77, 1997; Sevick-Muraca et al., Photochem. Photobiol., 66:55-64, 1997). However, previously described DOT systems and/or image algorithms have not been useful to obtain three-dimensional quantitation of fluorescence in deep tissues in living mammals.
The invention is based on the discovery that in vivo fluorochrome signals from specific targeted molecular probes, e.g., probes targeted for specific enzyme activities or DNA sequences, can be localized in three dimensions in deep tissues and be quantitated with high sensitivity using a specially designed imaging system for this purpose and relying on self-calibrated image reconstruction, and new algorithms to extract molecular maps.
In general, the invention features a near-infrared, fluorescence-mediated molecular tomography (FMT) imaging system that includes a NIR light source to provide incident light; a multipoint incident illumination array to direct light into an object, e.g., an animal or human patient, from two or more separate excitation points; multiple optic fibers to transmit light from the light source to each point in the multipoint incident illumination array; a multipoint detection array to collect light, e.g., fluorescent light, emitted from the object from two or more separate collection points; a two-dimensional emitted light array to transmit light emitted from the object to a detector; multiple optic fibers to transmit light from each collection point to a corresponding point on the two-dimensional emitted light array; and a detector to detect and convert light emitted from each point of the two-dimensional emitted light array into a digital signal corresponding to the light emitted from the object.
In this system, the emitted light can be continuous wave (CW) light, time-resolved (TR) light, or both CW and TR light.
The system can further include a processor that processes the digital signal produced by the detector to provide an image on an output device. The output device can provide multiple images simultaneously. The processor can be programmed to process the digital signal by i) generating a corrected fluorescence measurement by subtracting a background signal and filter bleed-through signal from collected fluorescence measurements; ii) generating a corrected intrinsic signal measurement by subtracting a background ambient light signal from collected intrinsic signal measurements; iii) generating a self-calibrated fluorescence measurement by dividing the corrected fluorescence measurement by the corrected intrinsic measurement; iv) generating a corrected background-medium diffuse signal by subtracting the collected background ambient light signal from a collected diffuse signal; and v) generating a self-calibrated intrinsic measurement by dividing the corrected intrinsic signal measurement by the corrected background-medium diffuse signal.
In other embodiments, the processor can be programmed to process the digital signal by i) generating a self-calibrated measurement M=M1xe2x88x92M3/M2xe2x88x92M4, wherein M1 is an emission wavelength fluorescence signal, M2 is an intrinsic signal, M3 is a background bleed-through signal, M4 is a background ambient light signal; ii) generating a self-calibrated intrinsic measurement Mxe2x80x2=log (M2xe2x88x92M4)/(M5xe2x88x92M4), wherein M5 is a background-medium diffuse signal; iii) minimizing a function F(U)=(Mxe2x88x92P xc3x97U)2 to obtain a distribution and magnitude of U, wherein U is a vector of unknown concentration of a target in the object being imaged and P is a forward predictor of M calculated by solving a diffusion equation for an appropriate geometry and background medium in fluorescence mode; iv) minimizing a function Fxe2x80x2(O)=(Mxe2x80x2xe2x88x92Pxe2x80x2xc3x97O)2 to obtain a distribution and magnitude of O; wherein O is a vector of unknown concentration of a fluorophore in the object, and Pxe2x80x2 is a forward predictor of Mxe2x80x2 calculated by solving a diffusion equation for the appropriate geometry and background medium in absorption/scattering mode; v) calculating an activation ratio AR=U/O; and vi) generating an image corresponding to AR.
The imaging system can include more than 100 optic fibers to transmit light into the patient and/or from each collection point of the detection array, and the detector array can include at least 100 collection points.
In this imaging system, the two-dimensional emitted light array can transmit to the detector a two-dimensional pattern of multiple points of light corresponding to light emitted from the patient in three-dimensions, wherein the pattern varies over time at a rate corresponding to switching of illumination from one to another of the two or more excitation points. In addition, the two or more excitation points are illuminated by the light source one at a time. In certain embodiments the NIR light directed into the object can be at a wavelength of from 550 to 950, e.g., 670 or 750 to 850, nanometers, and the detector can be a charge-coupled device (CCD) camera or include a photomultiplier tube.
The system can also include the NIR fluorescent (NIRF) molecular probes themselves. The probes can be activatable molecular probes.
The invention also features a method for displaying an optical molecular map corresponding to a ratio of a concentration of a molecular probe comprising a fluorophore administered to a patient to a concentration of an activated fluorophore corresponding to a specific target in the patient by: i) providing a first data set of fluorophore concentration based on intrinsic absorption; ii) providing a second data set of activated fluorophore concentration based on fluorescence; iii) dividing the first data set by the second data set on a point-by-point basis to provide a third data set; and iv) processing the third data set to provide an optical molecular map corresponding to a ratio of a concentration of a molecular probe comprising a fluorophore to a concentration of an activated fluorophore corresponding to a specific target in the patient.
In another aspect, the invention features a method of obtaining a three-dimensional, quantitative, molecular tomographic image of a target region within a patient, by administering a near-infrared fluorescent (NIRF) molecular probe to the patient, wherein the molecular probe selectively accumulates within a target region in the patient; directing near infrared light from multiple points into the patient; detecting fluorescent light emitted from the patient; and processing the detected light to provide a three-dimensional image that corresponds to the three-dimensional target region within the patient and to the quantity of molecular probe accumulated in the target region.
In this method, the three-dimensional image can be visualized on a two-dimensional output device. The processing can include digitizing the fluorescent signal emitted from the patient, self-calibrating the digital signal by combining fluorescent and intrinsic signal measurements from the patient and background medium, and reconstructing a three-dimensional, quantitative image. In certain embodiments, the processing includes i) generating a corrected fluorescence measurement by subtracting a background signal and filter bleed-through signal from collected fluorescence measurements; ii) generating a corrected intrinsic signal measurement by subtracting a background ambient light signal from collected intrinsic signal measurements; iii) generating a self-calibrated fluorescence measurement by dividing the corrected fluorescence measurement by the corrected intrinsic measurement; iv) generating a corrected background-medium diffuse signal by subtracting the collected background ambient light signal from a collected diffuse signal; and v) generating a self-calibrated intrinsic measurement by dividing the corrected intrinsic signal measurement by the corrected background-medium diffuse signal.
The processing can also include i) generating a self-calibrated measurement M=M1xe2x88x92M3/M2xe2x88x92M4, wherein M1 is an emission wavelength fluorescence signal, M2 is an intrinsic signal, M3 is a background bleed-through signal, M4 is a background ambient light signal; ii) generating a self-calibrated measurement Mxe2x80x2=log (M2xe2x88x92M4)/(M5xe2x88x92M4), wherein M5 is a background-medium diffuse signal; iii) minimizing a function F(U)=(Mxe2x88x92Pxc3x97U)2 to obtain a distribution and magnitude of U, wherein U is a vector of unknown concentration of a target in the object being imaged and P is a forward predictor of M calculated by solving a diffusion equation for an appropriate geometry and background medium in fluorescence mode; iv) minimizing a function Fxe2x80x2(O)=(Mxe2x80x2xe2x88x92Pxe2x80x2xc3x97O)2 to obtain a distribution and magnitude of O; wherein O is a vector of unknown concentration of a fluorophore in the object, and Pxe2x80x2 is a forward predictor of Mxe2x80x2 calculated by solving a diffusion equation for the appropriate geometry and background medium in absorption/scattering mode; v) calculating an activation ratio AR=U/O; and vi) generating an image corresponding to AR.
In these methods, the molecular probes can be administered systemically or locally by injecting a molecular probe, e.g., an activatable probe. The molecular probe can be locally injected into the target region or into a non-target region, for example, by intraperitoenal administration with systemic absorption and administration by an implanted slow-release compound or device such as a pump.
In certain embodiments of the new methods, the NIR light can be directed into the patient from at least 32 separate points of light arranged in a fixed three-dimensional geometry, or with a multipoint incident illumination array comprising a belt having at least 12 points of light. In addition, the spatial localizations of the multipoint incident illumination array and the multipoint detector array can be determined by image co-registration. In other embodiments, photon pulses are directed into the patient and the arrival of photons emitted from the patient is time-resolved using a separate array of photon detectors.
The emitted fluorescent light in these methods can be continuous wave (CW) light, time-resolved (TR) light, or both CW and TR light. In addition, the methods can be performed dynamically as function of time, and the image can be co-registered with an image obtained by magnetic resonance or computed tomography imaging. The multipoint incident illumination array (or detector array) can include a fiducial, and wherein the fiducial is used to determine the spatial localization of the array on the object.
The invention also features a method of detecting a cellular abnormality in a patient by using molecular probes targeted to a particular cellular abnormality, e.g., associated with a disease such as cancer, a cardiovascular disease, AIDS, a neurodegenerative disease, an inflammatory disease, or an immunologic disease. The invention also features a method of assessing the effect of a compound on a specified molecular target by using a molecular probe that is activated by the molecular target, wherein the probe is contacted to the target, the target is imaged prior to and after contact with the molecular probe, and the corresponding images are compared, wherein a change in the molecular target indicates the compound is effective. For example, the specified molecular target can be a protease, and the compound can be a protease inhibitor.
A molecular probe is a probe that is targeted to a molecular structure, such as a cell-surface receptor or antigen, an enzyme within a cell, or a specific nucleic acid, e.g., DNA, to which the probe hybridizes. A fluorophore is an agent that fluoresces. A fluorochrome is an agent that fluoresces (e.g., a fluorophore) and has a color.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The new methods and systems provide various advantages. For example, the new methods and systems provide for the first time the ability to detect fluorescence activation, e.g., by enzyme activation, in deep tissue and to provide localization and quantitation in three dimensions. In addition, the new methods provide non-invasive, molecular imaging to provide information at subcellular levels.
The impact of the new molecular imaging techniques is potentially enormous. First, the new methods and systems can provide insight into specific molecular abnormalities that form the basis of many diseases, e.g. up-regulated proteases, other enzymes, cell surface receptors, cyclins, cytokines or growth factors in cancer. Second, the new methods can be used to assess efficacy of novel targeted therapies at a molecular level, long before phenotypic changes occur. This, in turn, is expected to have an impact in drug development, drug testing, and choosing appropriate therapies and therapy changes in a given patient. Third, the new molecular imaging/quantitation methods and systems potentially enable one to study the genesis of diseases in the intact microenvironment of living systems. Fourth, the new methods of fluorescence-mediated molecular tomographic imaging are useful for testing novel drug delivery strategies. Fifth, the imaging methods allow one to gain three-dimensional information that is much faster to obtain than is currently possible with time consuming and labor intensive conventional, basic science techniques.
The new imaging systems and methods will have broad applications in a wide variety of novel biologic, immunologic, and molecular therapies designed to promote the control and eradication of numerous different diseases including cancer, cardiovascular, neurodegenerative, inflammatory, infectious, and other diseases. Furthermore, the described detection systems and methods will have broad applications for seamless disease detection and treatment in combined settings.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.